In Situ Enzyme Immobilization by Covalent Organic Frameworks

Enzyme immobilization is a widely reported method to favor the applicability of enzymes by enhancing their stability and re-usability. Among the various existing solid supports and immobilization strategies, the in situ encapsulation of enzymes within crystalline porous matrices is a powerful tool to design biohybrids with a stable and protected catalytic activity. However, to date, only a few metal–organic frameworks (MOFs) and hydrogen-bonded organic frameworks (HOFs) have been reported. Excitingly, for the first time, Y. Chen and co-workers expanded the in situ bio-encapsulation to a new class of crystalline porous materials, namely covalent organic frameworks (COFs). The enzyme@COF materials not only exhibited high enzyme loading with minimal leaching, high catalytic activity and selectivity, chemical and long-term stability and recyclability but could also be scaled up to a few grams. Undoubtedly, this work opens new striking opportunities for enzymatic immobilization and will stimulate new research on COF-based matrices.     

Sol-Gel Entrapment of Escherichia coli

Whole cell bacteria have been entrapped within sol-gel silica matrices in order to perform bio-catalytic experiments. Escherichia coli have been chosen as a model for sol-gel encapsulation. Transmission electron microscopy shows that bacteria are randomly dispersed within the silica matrix and that their cellular organization is preserved. The -galactosidase activity of entrapped E. coli was studied using p-NPG as a substrate. The formation of p-nitrophenol was followed by optical absorption. These experiments show that E. coli still exhibit noticeable enzymatic activity after encapsulation in wet gels. They follow the well known Michaelis-Menten kinetic law but their activity decreases in dried xerogels.     

Sequential pressurized liquid extraction to determine brain-originating fatty acids in meat products as markers in bovine spongiform encephalopathy risk assessment studies

A new approach using sequential pressurized liquid extraction described recently [J. Poerschmann, R. Carlson, J. Chromatogr. A, 1127 (2006) 18-25] was applied to determine lipid markers originating from central nervous system (CNS) tissue of cows in heat-processed sausages. These studies are very important in quality control as well as risk assessment studies in the face of the bovine spongiform encephalopathy (BSE) crisis. Diagnostic CNS lipid markers, which should not be present in meat products without CNS addition, were recognized on complete transesterification as polar 2-hydroxy-fatty acids (2OH-24:0, 2OH-24:1, 2OH-22:0, 2OH-18:0, shorthand designation) as well as odd-numbered non-branched fatty acids beyond C(22). An array of other fatty acids including lignoceric acid (24:0), nervonic acid (24:1), arachidonic acid (20:4), and polyunsaturated nC(22)-surrogates are strongly related to CNS lipids, but occur as traces in meat products without CNS addition as well, thus reducing their value as diagnostic markers. Samples including meat products without CNS addition, meat with 3% CNS addition, as well as pure CNS homogenates, were subjected to sequential PLE (pressurized liquid extraction) consisting of two steps: n-hexane/acetone 9:1 (v/v) extraction at 50 degrees C to remove neutral lipids, followed by chloroform/methanol 1:4 (v/v) extraction at 110 degrees C to isolate polar CNS lipids (two 10 min PLE cycles each). To enhance the fractionation efficiency, cyanopropyl modified silica as well as chemically not modified silica sorbent was used at the outlet of the PLE cartridge to retard polar lipids in the first extraction step. This method proved superior to widely distributed exhaustive lipid extraction followed by solid-phase extraction (SPE) using silica regarding lipid recoveries and clear-cut boundaries between lipid classes. Methodological studies showed that the alcoholysis using trimethylchlorosilane/methanol (1:9, v/v) is an excellent method for the complete transesterification of lipids and quantitative formation of methyl esters.     

Enzyme-functionalized mesoporous silica for bioanalytical applications

The unique properties of mesoporous silica materials (MPs) have attracted substantial interest for use as enzyme-immobilization matrices. These features include high surface area, chemical, thermal, and mechanical stability, highly uniform pore distribution and tunable pore size, high adsorption capacity, and an ordered porous network for free diffusion of substrates and reaction products. Research demonstrated that enzymes encapsulated or entrapped in MPs retain their biocatalytic activity and are more stable than enzymes in solution. This review discusses recent advances in the study and use of mesoporous silica for enzyme immobilization and application in biosensor technology. Different types of MPs, their morphological and structural characteristics, and strategies used for their functionalization with enzymes are discussed. Finally, prospective and potential benefits of these materials for bioanalytical applications and biosensor technology are also presented. Figure Enzyme-functionalized mesoporous silica fibers and their integration in a biosensor design. The immobilization process takes place essentially in the silica micropores.     

Loading of silica spheres on concaved CuS cuboctahedrons using 3-aminopropyltrimethoxysilane as a linker

A new composite structure made of unique CuS cuboctahedron crystals and coating silica spheres with diameters ranged from 30 to 730 nm has been demonstrated. 3-aminopropyltrimethoxysilane acted as a linker, which provided hydrogen bonding and metal-ligand bonding interaction with the facets of CuS crystals and covalent bonding of its functional silane groups onto the surface of hydrophilic silica spheres, resulting in the formation of such a CuS/silica composite structure. The suitable proportion of the amount of silica spheres and CuS crystals determined the perfection of the CuS crystals/silica composite. The successful loading of silica spheres onto the polar facets of unique CuS cuboctahedron crystals represents a new way to prepare novel composite materials with special shape.     

New fractionation scheme for lipid classes based on "in-cell fractionation" using sequential pressurized liquid extraction

A new preparation scheme is proposed to fractionate neutral lipids (acylglycerines, sterol esters, long-chain free fatty acids) from polar phospholipids in biological matrices. This fractionation is mandatory in the microbial community, for the control of bioremediation processes, in the study of phytoplankton growth in lakes and rivers, and in the quality control of processed food. Basically, a two-step pressurized liquid extraction (PLE) scheme is combined with an "in-cell-fractionation" using silica-based sorbents placed at the outlet of the PLE cartridge. The optimized extraction scheme consists of n-hexane/acetone (9:1, v/v) extraction at 50 degrees C (2 cycles, 10 min each) to obtain neutral lipids followed by chloroform/methanol (1:4, v/v) extraction at 110 degrees C (2 cycles, 10 min each). Thermally pre-treated silicic acid and cyanoproyl-modified silica turned out to be appropriate sorbents to ensure clear-cut boundaries between neutral lipids and phospholipids. The proposed protocol is superior to commonly used approaches consisting of an exhaustive lipid extraction followed by off-line lipid fractionation using solid-phase extraction (SPE) regarding fractionation efficiency, time and solvent consumption. In this paper, it is also shown that the transmethylation using trimethylchlorosilane/methanol (1:9, v/v) provides a complete reaction to give methyl esters without artefact formation across the array of different lipid classes even with polyunsaturated fatty acid moieties.     

Preparation and characterization of salt hydrates encapsulated in polyamide membranes

Thermodynamic water activity control is a common technique in organic-phase biocatalysis. This can be accomplished by using the transitions of salt hydrates between their various hydrated forms as a water buffer. While this technique is well established, the use of free salt crystals in the reaction mixture poses numerous problems such as difficult recovery and poisoning of the biocatalyst. This article outlines a novel technique for the encapsulation of such materials which avoids these difficulties. The characterization of the capsules and their use as water activity buffers has also been described. Hydrates of Na₂HPO₄ were encapsulated in a polyamide membrane by interfacial polycondensation (IPC) of sebacoyl dichloride and diethylene triamine soaked onto the surface of the salt crystals. This technique, non-aqueous interfacial polycondensation (NAIPC) circumvents the need for the use of an aqueous phase to supply the polar reactant, the amine, thereby facilitating the encapsulation of water soluble materials. The coatings thus produced have an asymmetric membrane-like structure. A thin, non-porous, layer around the salt crystal supports a superstructure of porous polymer. This composite structure facilitates diffusion of material through the capsule wall and the use of hydrophilic polyamides for encapsulation promotes the transport of water. The capsules produced were between 0.5 and 2.5 mm in size and were of adequate mechanical strength to withstand osmotic pressure differences upto 26 bar and resist attritive forces experienced during their use.     

Polysiloxane-encapsulated stationary phase for reversed-phase high-performance liquid chromatography

Summary Silica beads of 6-μm average diameter were silanized with methylvinyldiethoxysilane and then subjected to encapsulation with poly(methylvinylsiloxane). The resulting product is a new stationary phase for reversed-phase high performance liquid chromatography (RP-HPLC) which has superior ability for the separation of polar, non-polar and basic compounds. The chromatographic peaks are symmetric. Its stability has been studied; after continuous use for three months the carbon content and chromatographic behaviour of the phase were unchanged. on to the silica surface to given an uniform organic film. Material prepared in this way has both good chromatographic behaviour and superior selectivity. Because contact of the silica matrix with the mobile phase is avoided, the alkali-resisting ability of the stationary phase is increased. The non-specific adsorption of alkaline solutes on to the silica surface is also avoided because of the complete coverage of surface silanol groups. Reports of stationary phases encapsulated with polystyrene [6], polybutadiene [I] and octadecylsiloxane polymers have recently appeared in the literature [3]. In this paper we report the encapsulation of poly-(methylvinylsiloxane) (analogous to the phase SE-31 often used in GC) on to a silica matrix previously modified with methylvinyldiethoxysilane. The resulting phase has superior performance in reversed-phase HPLC.     

Trends in structuring edible emulsions with Pickering fat crystals

The pace of development of edible Pickering emulsions has recently soared, as interest in their potential for texture modification, calorie reduction and bioactive compound encapsulation and delivery has risen. In the broadest sense, Pickering emulsions are defined as those stabilized by interfacially-adsorbed solid particles that retard and ideally prevent emulsion coalescence and phase separation. Numerous fat-based species have been explored for their propensity to stabilize edible emulsions, including triglyceride and surfactant-based crystals and solid lipid nanoparticles. This review explores three classes of fat-based Pickering stabilizers, and proposes a microstructure-based nomenclature to delineate them: Type I (surfactant-mediated interfacial crystallization), Type II (interfacially-adsorbed nano- or microparticles) and Type III (shear-crystallized droplet encapsulation matrices). Far from simply reporting the latest findings on these modes of stabilization, challenges associated with these are also highlighted. Finally, though emphasis is placed on food emulsions, the fundamental precepts herein described are equally applicable to non-food multicomponent emulsion systems.     

复杂样品痕量极性小分子化合物的样品前处理及分析方法研究进展

核苷、胺、氨基酸等极性小分子化合物是生物、食品、环境等领域重要的研究对象,但各种复杂基体中痕量极性小分子的分离分析需要高效的前处理介质和技术以及快速灵敏的分析方法。本文综述了硅胶材料、有机聚合物、炭材料和硼酸材料等样品前处理分离介质及反相液相色谱、亲水作用色谱等分析方法在复杂样品痕量极性小分子化合物分析中的应用,并展望了其发展趋势。     

Liquid crystals and biological morphogenesis: Ancient and new questions

Superposed series of arcs were observed in extracellular matrices of diverse tissues in plants and animals, and also within certain chromosomes. These bow-shaped patterns were shown to originate from oblique sections in a three-dimensional arrangement of fibrils, which was reminiscent of a well-known structure present in a specific type of liquid crystal, called “cholesteric”, since it was first described in several cholesterol esters. These liquid crystals were also obtained with numerous chiral polymers, such as double-stranded nucleic acids, diverse polypeptides and polysaccharides. However, the fibrous matrices with arced patterns are not at all fluid in general and are considered as stabilized analogues of cholesteric liquid crystals. These geometrical similarities between fibrous tissues and liquid crystals were at the origin of numerous works defining a new type of self-assembly involving two successive steps: a phase transition from an isotropic liquid phase to a liquid crystalline one, followed by a sol–gel stabilization of the liquid crystal. One is tempted to apply to these biological materials the main concepts introduced by physicists in the field of liquid crystals, but many aspects require a new discussion, especially because living cells are present in such systems, and this opens considerable perspectives for research.     

Poly(ethylene glycol)/β-cyclodextrin covalent gel networks: host matrices for studying radical processes in plant extract–riboflavin systems following UV irradiation

Finding new, biocompatible matrices that allow us to model the generation of free radicals is of utmost importance for balancing the harmful and beneficial effects of the latter. In this respect, we report here the simultaneous encapsulation of the radical source and the antioxidant agent in a polyethylene glycol/β-cyclodextrin (PEG/β-CD) covalent gel network. We used electron paramagnetic resonance spectroscopy to evaluate the scavenging action of plant extracts (purple loosestrife, comfrey, milfoil, horsetail, thyme, carob, green coffee) embedded in PEG/β-CD gels. Free radicals were generated in situ by UV irradiation of riboflavin co-embedded in the gels. Prior to this, the extracts were characterized in what concerns their antioxidant activity, and their major polyphenolic constituents were quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry. Purple loosestrife showed the highest antioxidant capacity, followed by comfrey and milfoil. Using the 5,5-dimethyl-1-pyrroline N-oxide spin trap, we have demonstrated that the gel-embedded extracts effectively scavenge the reactive carbon-centered free radicals generated in gel. The PEG/β-CD gels have been shown to be a valuable alternative matrix for the encapsulation of plant active principles having antioxidant activity. Moreover, co-encapsulation of the radical source transforms these gels into a controlled environment in which free radical processes can be tailored.     

Sub-critical fluid extraction of morphinic alkaloids in urine and other liquid matrices after adsorption on solid supports

Summary Supercritical fluid extraction (SFE) of liquid matrices is difficult and requires special instrumentation. An alternative is to load the liquid on a solid support prior to extraction. This procedure yields higher selectivity and can be used as a clean-up step when very complex liquid matrices, such as urine, are to be analyzed. This paper demonstrates the use of various solid supports in order to extract morphinic alkaloids with subcritical fluids (CO₂ + polar modifier). Optimization of the procedure with regard to loading conditions, SFE parameters such as pressure, extraction time, flow rate and eluent composition, was carried out with morphinic alkaloids in aqueous solution. As a real-life application, SFE of opiates in urine were tested with two silica bonded phase supports (C18 (polygosil 60-4063, from Macherey-Nagel) and Bond Elut Certify from Analytichem International). Recovery, variation coefficients and linearity are described for each procedure. Bond Elut Certify silica was chosen as the best solid support and SFE is compared with conventional solid phase extraction.     

Silica modified with a thiourea derivative as a new stationary phase for hydrophilic interaction liquid chromatography

Nowadays there are limited types of commercially available stationary phases for hydrophilic interaction liquid chromatography and therefore new ones with unique selectivity are urgently in demand to meet the need of separations of various polar and hydrophilic analytes. The present study describes the preparation and evaluation of a new stationary phase based on thiourea derivative modified silica for hydrophilic interaction liquid chromatography. Thiourea derivative was bonded onto the surface of silica particles via a mild addition reaction between –NH₂ and –SCN, and the result of elemental analysis together with infrared analysis and solid‐state NMR spectroscopy proved that the synthesis method was feasible. The new stationary phase succeeded in fast separations of a wide range of polar and hydrophilic analytes and exhibited excellent separation performance, especially unique selectivity. Furthermore, the effects of water content, buffer pH, and salt concentration on retention indicated that a complicated separation mechanism rather than partitioning was involved in the stationary phase and hydrogen bonding interaction between analytes and thiourea functional group could play a very important role in its selectivity. For sure, the new stationary phase is of a great potential as a new type of hydrophilic interaction liquid chromatographic stationary phase.     

Comparative chromatography of lectins and bioactivity recovery of the immunologic hormone leukoregulin on derivatized silica and on cross-linked agarose molecular sizing high-performance liquid chromatographic matrices

This investigation compares the performance of the new zirconia stabilized silica and cross-linked agarose size-exclusion matrices to Spherogel-TSK 3000 SWG silica in high-performance liquid chromatographic separation of proteins possessing a range of molecular weights present in many lymphokine preparations and in recovery of bioactivity as measured by leukoregulin proliferation inhibitory activity. Retention time versus log molecular weight of protein standards was linear from 12,500 to 290,000 on the agarose and from 32,000 to 290,000 on the other columns. Recovery of leukoregulin proliferation inhibitory activity directed against RPMI 2650 epidermoid carcinoma cells was 90% from the silica, 88% from the agarose and 35% from the zirconia stabilized silica columns.     

Formation of colloid crystals from polymer-modified monodisperse colloidal silica in organic solvents

The formation of colloid crystals from monodisperse and polymer-modified silica particles in organic solvents was investigated. Maleic anhydride–styrene copolymer-modified silica formed crystals in polar organic solvents, which dissolve the copolymer, while the original colloidal silica formed crystals in organic solvents which were miscible with water. The critical volume fraction in the crystal formation of the polymer-modified silica was lower than that from the unmodified silica in the same solvent. Polystyrene- and poly(methyl methacrylate)-modified silica particles also crystallized in organic solvents, but the features of the formation were different from those of poly(maleic anhydride-styrene)-modified particles. Received: 19 September 1998 Accepted in revised form: 1 January 1999     

Liquid-crystalline phases of circular superhelical plasmid DNA and their modification by the action of nuclease enzymes

CD spectra of liquid-crystalline dispersions, X-ray diffraction patterns and optical textures of liquid crystals prepared from native superhelical DNA in poly(ethyleneglycol)-containing water-salt solutions before and after treatment of DNA with micrococcal nuclease have been obtained. It was found that condensation of native superhelical DNA is accompanied by the formation of liquid crystals with a non-specific optical texture. After treatment of the DNA, liquid-crystalline dispersions, with Micrococcal nuclease the DNA is able to form two similar types of liquid crystals with abnormal optical activity which differ in the peculiarities of their textures. The data obtained demonstrate the formation of multiple types of liquid crystals from high molar mass double-stranded optically active DNA molecules.     

非传统型液晶分子设计与工程研究进展

从液晶基元连接方式、液晶分子拓扑结构以及凝聚态自组织方式等方面扼要介绍和评述了非传统型液晶分子设计与工程研究进展,并重点介绍了可望引起液晶显示技术革命的双轴向列相香蕉形液晶研究的突破性工作,展望了非传统型液晶分子设计和复杂自组织超分子液晶领域今后的发展方向。     

Effect of surface on the thermodynamic properties of polar liquids and liquid crystals trapped in a porous medium

The heat capacity of some polar liquids (2,6-lutidine, aniline and nitrobenzene) and liquid crystals (nitrophenyldodecyloxybenzoate and dodecylcyanobiphenyl) trapped in glass and nickel porous matrices have been measured by using a high-resolution adiabatic calorimeter. A decrease in the molar heat capacity of substances trapped in porous media compared with the bulk molar heat capacity was observed. A new phenomenon caused by the effect of the glass surface on the confined fluid was revealed. This phenomenon was manifested as an unusual behaviour of the heat capacity versus temperature curve (a ‘crevasse’ in a curve). In addition, we observed the nematic phase of dodecylcyanobiphenyl liquid crystal induced by the variable curvature of the glass matrix surface.     

Helical Nano‐crystallite (HNC) Phases: Chirality Synchronization of Achiral Bent‐Core Mesogens in a New Type of Dark Conglomerates

Spontaneous generation of macroscopic homochirality in soft matter systems by self‐assembly of exclusively achiral molecules under achiral conditions is a challenging task with relevance for fundamental scientific research and technological applications. Dark conglomerate phases (DC phases), being optically isotropic mesophases composed of conglomerates of macroscopic chiral domains and formed by some non‐chiral bent‐core mesogens, represent such a case. Here we report two new series of non‐symmetric bent‐core molecules capable of forming a new type of mirror symmetry broken DC phases. In the synthesized molecules, a bent 4‐bromoresorcinol core is connected to a phenyl benzoate wing and an azobenzene wing with or without additional peripheral fluorine substitution. The self‐assembly was investigated by DSC, polarizing microscopy, electro‐optical studies and XRD. Chiral and apparently achiral DC phases were observed besides distinct types of lamellar liquid crystalline phases with different degree of polar order, allowing the investigation of the transition from smectic to DC phases. This indicates a process in which increased packing density at first gives rise to restricted rotation and thus to growing polar order, which then leads to chirality synchronization, layer frustration and nano‐scale crystallization. Topological constraints arising from the twisted packing of helical conformers in lamellar crystals is proposed to lead to amorphous solids composed of helical nano‐crystallites with short coherence length (HNC phases). This is considered as a third major type of DC phases, distinct from the previously known liquid crystalline sponge phases and the helical nano‐filament phases (HNF phases). Guidelines for the molecular design of new materials capable of self‐assembly into these three types of DC phases are proposed.